Claims for Patent: 8,637,306

Summary for Patent: 8,637,306

Title:	Production of carrier-peptide conjugates using chemically reactive unnatural amino acids
Abstract:	Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.
Inventor(s):	Young; Travis (San Diego, CA), Schultz; Peter G. (La Jolla, CA)
Assignee:	The Scripps Research Institute (La Jolla, CA)
Application Number:	12/653,274
Patent Claims:	1. A biologically and/or pharmacologically active carrier polypeptide-target polypeptide conjugate for therapeutic use, produced by a method comprising: incorporating a first unnatural amino acid residue comprising a first reactive group into a carrier polypeptide during translation of the carrier polypeptide in a methylotrophic yeast cell; incorporating a second unnatural amino acid residue comprising a second reactive group into a therapeutic target polypeptide during synthesis or translation of the target polypeptide, wherein the target polypeptide is different from the carrier polypeptide, wherein the first and second reactive groups are different and are selected from a keto group and an aminooxy group; wherein incorporating the first unnatural amino acid into the carrier polypeptide during translation comprises: (a) providing a methylotrophic yeast cell comprising (i) the first unnatural amino acid, (ii) an orthogonal tRNA-synthetase (O-RS) derived from Escherichia coli, wherein providing an O-RS comprises providing an O-RS polynucleotide under the transcriptional control of a FLD1 promoter; (iii) an orthogonal tRNA (O-tRNA) derived from Escherichia coli, wherein the O-RS and the O-tRNA are encoded in a cassette on a single plasmid and stably integrated into the cell genome at an ARG4 gene, and wherein the O-RS preferentially aminoacylates the O-tRNA with the first unnatural amino acid in the methylotrophic yeast cell; and (iv) a nucleic acid encoding the carrier polypeptide, wherein the nucleic acid comprises a selector codon that is recognized by the O-tRNA, and wherein the nucleic acid is encoded in a cassette on a second plasmid and stably integrated into the cell genome; and, (b) translating the nucleic acid, thereby incorporating the first unnatural amino acid into the carrier polypeptide during translation in the methylotrophic yeast cell; and, reacting the first and second unnatural amino acid residues via in vitro oxime ligation to produce the covalently coupled carrier polypeptide-target polypeptide conjugate. 2. A biologically and/or pharmacologically active carrier polypeptide-target polypeptide conjugate for therapeutic use, comprising: a carrier polypeptide domain comprising a first unnatural amino acid residue; and, a therapeutic target polypeptide domain comprising a second unnatural amino acid residue, wherein the carrier polypeptide domain and target polypeptide domain are conjugated together through the first and second unnatural amino acid residues via in vitro oxime ligation of reactive groups in the first and second residues, wherein the reactive groups are different and are selected from a keto group and an aminooxy group; wherein the target polypeptide is different from the carrier polypeptide, wherein the second unnatural amino acid is different from the first unnatural amino acid, and wherein the carrier polypeptide is produced during translation in a methylotrophic yeast cell; wherein incorporating the first unnatural amino acid into the carrier polypeptide during translation comprises: (a) providing a methylotrophic yeast cell comprising (i) the first unnatural amino acid, (ii) an orthogonal tRNA-synthetase (O-RS) derived from Escherichia coli, wherein providing an O-RS comprises providing an O-RS polynucleotide under the transcriptional control of a FLD1 promoter; (iii) an orthogonal tRNA (O-tRNA) derived from Escherichia coli, wherein the O-RS and the O-tRNA are encoded in a cassette on a single plasmid and stably integrated into the cell genome at an ARG4 gene, and wherein the O-RS preferentially aminoacylates the O-tRNA with the first unnatural amino acid in the methylotrophic yeast cell; and (iv) a nucleic acid encoding the carrier polypeptide, wherein the nucleic acid comprises a selector codon that is recognized by the O-tRNA, and wherein the nucleic acid is encoded in a cassette on a second plasmid and stably integrated into the cell genome; and, (b) translating the nucleic acid, thereby incorporating the first unnatural amino acid into the carrier polypeptide during translation in the methylotrophic yeast cell. 3. A cell comprising the carrier polypeptide-target polypeptide conjugate of claim 2. 4. The conjugate of claim 1, wherein the carrier or target polypeptide is produced in a Candida cell, a Hansenula cell, a Pichia cell, or a Torulopsis cell. 5. The conjugate of claim 1, wherein the carrier polypeptide is homologous to a human serum albumin (HSA). 6. The conjugate of claim 1, wherein the carrier polypeptide is or is homologous to an antibody, a HER2 antibody, and OKT3 antibody, an antibody fragment, an Fab, an Fc, an scFv, an albumin, a serum albumin, a bovine serum albumin, an ovalbumin, a c-reactive protein, a conalbumin, a lactalbumin, a keyhole limpet hemocyanin (KLH), an ion carrier protein, an acyl carrier protein, a signal transducing adaptor protein, an androgen-binding protein, a calcium-binding protein, a calmodulin-binding protein, a ceruloplasmin, a cholesterol ester transfer protein, an f-box protein, a fatty acid-binding proteins, a follistatin, a follistatin-related protein, a GTP-binding protein, an insulin-like growth factor binding protein, an iron-binding protein, a latent TGF-beta binding protein, a light-harvesting protein complex, a lymphocyte antigen, a membrane transport protein, a neurophysin, a periplasmic binding protein, a phosphate-binding protein, a phosphatidylethanolamine binding protein, a phospholipid transfer protein, a retinol-binding protein, an RNA-binding protein, an s-phase kinase-associated protein, a sex hormone-binding globulin, a thyroxine-binding protein, a transcobalamin, a transcortin, a transferrin-binding protein, or a vitamin D-binding protein. 7. The conjugate of claim 1, wherein the target polypeptide is or is homologous to a TSP-1, an ABT-510, a glugacon-like peptide-1 (GLP-1), a parathyroid hormone (PTH), a ribosome inactivating protein (RIP), an angiostatin, an Exedin-4, an apoprotein, an atrial natriuretic factor, an atrial natriuretic polypeptide, an atrial peptide, a C-X-C chemokine, a T39765, a NAP-2, an ENA-78, a gro-a, a gro-b, a gro-c, an IP-10, a GCP-2 , a NAP-4, an a PF4, a MIG, a calcitonin, a c-kit ligand, a cytokine, a CC chemokine, a monocyte chemoattractant protein-1, a monocyte chemoattractant protein-2, a monocyte chemoattractant protein-3, a monocyte inflammatory protein-1 alpha, a monocyte inflammatory protein-1 beta, a RANTES, an I309, an R83915, an R91733, a T58847, a D31065, a T64262, a CD40 ligand, a complement inhibitor, a cytokine, an epithelial neutrophil activating peptide-78, a GRO'.UPSILON., a MGSA, a GRO.beta., a GRO.gamma., a MIP1-.alpha., a MIP1-.beta., an MCP-1, an epithelial neutrophil activating peptide, an erythropoietin (EPO), an exfoliating toxin, a fibroblast growth factor (FGF), an FGF21, a G-CSF, a gonadotropin, a growth factor, a Hirudin, an LFA-1, a human insulin, a human insulin-like growth factor (hIGF), an hIGF-I, an hIGF-II, a human interferon, an IFN-.alpha., an IFN-.beta., an IFN-.gamma., an interleukin, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, a keratinocyte growth factor (KGF), a leukemia inhibitory factor, a neurturin, a PDGF, a peptide hormone, a pleiotropin, a pyrogenic exotoxin A, a pyrogenic exotoxin B, a pyrogenic exotoxin C, a relaxin, a somatostatin, a superoxide dismutase, a thymosin alpha 1, a human tumor necrosis factor (hTNF), a human tumor necrosis factor alpha, a human tumor necrosis factor beta, a Ras, a Tat, an inflammatory molecule, a signal transduction molecule, a bovine pancreatic trypsin inhibitor (BPTI), or a BP320 antigen. 8. The conjugate of claim 1, wherein incorporating the first unnatural amino acid into the carrier polypeptide results in an HSA variant comprising the first unnatural amino acid. 9. The conjugate of claim 8, wherein the first unnatural amino acid is a p-acetylphenylalanine. 10. The conjugate of claim 9, wherein the first unnatural amino acid is incorporated into the HSA variant at amino acid position 37, wherein numbering of amino acid position is relative to that of SEQ ID NO: 1. 11. The conjugate of claim 9, wherein the first unnatural amino acid is incorporated into the HSA variant during translation. 12. The conjugate of claim 1, wherein incorporating the second unnatural amino acid into the target polypeptide results in a TSP-1 variant comprising the second unnatural amino acid. 13. The conjugate of claim 1, wherein incorporating the second unnatural amino acid into the target polypeptide results in an ABT-510 variant comprising the second unnatural amino acid. 14. The conjugate of claim 12 or 13, wherein the second unnatural amino acid is an .epsilon.-(2-(aminooxy)acetyl)-L-lysine. 15. The conjugate of claim 14, wherein the .epsilon.-(2-(aminooxy)acetyl)-L-lysine is incorporated into the TSP-1 variant at amino acid position 6 wherein numbering of amino acid position is relative to that of SEQ ID NO: 2. 16. The conjugate of claim 14, wherein the .epsilon.-(2-(aminooxy)acetyl)-L-lysine is incorporated into the TSP-1 variant at amino acid position 1 wherein numbering of amino acid position is relative to that of SEQ ID NO: 2. 17. The conjugate of claim 14, wherein the .epsilon.-(2-(aminooxy) acetyl)-L-lysine is incorporated into the TSP-1 variant during synthesis. 18. The conjugate of claim 14, wherein the .epsilon.-(2-(aminooxy) acetyl)-L-lysine is incorporated into the ABT-510 variant at amino acid position 6 wherein numbering of amino acid position is relative to that of SEQ ID NO: 3. 19. The conjugate of claim 14, wherein the .epsilon.-(2-(aminooxy)acetyl)-L-lysine is incorporated into the ABT-510 variant at amino acid position 1 wherein numbering of amino acid position is relative to that of SEQ ID NO: 3. 20. The conjugate of claim 14, wherein the .epsilon.-(2-(aminooxy)acetyl)-L-lysine is incorporated into the ABT-510 variant during synthesis. 21. The conjugate of claim 1, wherein the carrier polypeptide is an HSA variant, wherein the first unnatural amino acid is a p-acetylphenylalanine, wherein the p-acetylphenylalanine is incorporated into an HSA variant at amino acid position 37 during translation, wherein the numbering of amino acid position in the HSA variant is relative to SEQ ID NO: 1, wherein the target polypeptide is a TSP-1 variant, wherein the second unnatural amino acid is .epsilon.-(2-(aminooxy)acetyl)-L-lysine, wherein the .epsilon.-(2-(aminooxy) acetyl)-L-lysine is incorporated into the TSP-1 variant at amino acid position 6 during synthesis, wherein the numbering of amino acid position in the TSP-1 variant is relative to SEQ ID NO: 2, and wherein the p-acetylphenylalanine and the .epsilon.-(2-(aminooxy) acetyl)-L-lysine are reacted via oxime ligation to produce an HSA-TSP-1conjugate. 22. The conjugate of claim 1, wherein the carrier polypeptide is an HSA variant, wherein the first unnatural amino acid is a p-acetylphenylalanine, wherein the p-acetylphenylalanine is incorporated into an HSA variant at amino acid position 37 during translation, wherein the numbering of amino acid position in the HSA variant is relative to SEQ ID NO: 1, wherein the target polypeptide is an ABT-510 variant, wherein the second unnatural amino acid is .epsilon.-(2-(aminooxy)acetyl)-L-lysine, wherein the .epsilon.-(2-(aminooxy) acetyl)-L-lysine is incorporated into the ABT-510 variant at amino acid position 6 during synthesis, wherein the numbering of amino acid position in the ABT-510 variant is relative to SEQ ID NO: 3, and wherein the p-acetylphenylalanine and the .epsilon.-(2- (aminooxy) acetyl)-L-lysine are reacted via oxime ligation to produce an HSA-ABT-510conjugate. 23. The conjugate of claim 1, wherein said oxime ligation reaction is greater than 50% efficient, greater than 70% efficient, or greater than 90% efficient.

Details for Patent 8,637,306

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Eli Lilly And Company	HUMULIN R U-100	insulin human	Injection	018780	10/28/1982		2028-12-10
Eli Lilly And Company	HUMULIN R U-500	insulin human	Injection	018780	12/29/2015		2028-12-10
Eli Lilly And Company	HUMULIN R U-100	insulin human	Injection	018780	08/06/1998		2028-12-10
Eli Lilly And Company	HUMULIN R U-500	insulin human	Injection	018780	03/31/1994		2028-12-10
Eli Lilly And Company	HUMULIN R U-100	insulin human	Injection	018780	05/25/2018		2028-12-10